α-玉米赤霉醇对肿瘤坏死因子α诱导的人内皮细胞活性氧产生及其激活信号通路的影响

Effect of α-zearalanol on the generation of reactive oxygen species （ROS） and ROS-activated signal transduction in tumor necrosis factor α-stimulated human endothelial cells

目的探讨α-玉米赤霉醇对肿瘤坏死因子α（TNF-α）刺激的人脐静脉内皮细胞中活性氧（ROS）产生及其对激活信号通路的影响。方法用小分子RNA干扰（siRNA）技术消除人脐静脉内皮细胞的NADPH氧化酶p47^phox亚基；用分子探针2，7-DCF测定细胞内ROS的产生量；用RT-PCR测定p47^phox mRNA的表达以及用细胞免疫组化方法测定p47^phox蛋白的表达；用Western印迹测定ERK2及核因子（NF）-κB、应激蛋白（SP-1）和活化因子蛋白的表达。结果TNF-α刺激使细胞内ROS的产生量较对照组高155，4％；α-玉米赤霉醇预处理能够剂量依赖地抑制TNF—α对ROS的诱导效应。TNF-α作用细胞24h后，p47^phox的mRNA表达水平较对照组高212．8％，蛋白的表达也明显高；α-玉米赤霉醇预处理使TNF-α诱导的p47^phox mRNA表达水平低63．0％，蛋白表达水平也明显下降。p47^phox的siRNA完全阻断TNF-α诱导ROS的产生。α-玉米赤霉醇预处理和p47^phox的siRNA均阻断TNF-α对细胞外信号调控激酶的激活和转录因子SP-1的核转位，明显地抑制了NF—κB的核转位。结论α-玉米赤霉醇能够抑制内皮细胞中TNF—α诱导的ROS的产生及由ROS激活的信号转导通路，主要机制是通过抑制NADPH氧化酶p47^phox亚基的表达。

Objective To investigate the effects of α-zearalanol （ct-ZAL） on the generation of reactive oxygen species （ROS） and ROS-activated signal transduction in the tumor necrosis factor（TNF-α）- stimulated human umbilical vein endothelial cells （HUVECs）. Methods HUVECs were cultured and divided into 4 groups： （1） normal control group, （2）TNF-α stimulated group, undergoing TNF-α stimulation for 24 h, （3） α-ZAL retreatment group, undergoing re-treatment withα-ZAL of the concentrations of 1 × 10^-8, 1 × 10^-7, or 1 × 10^-6 mol/L for 1 h, then stimulation of TNF-α for 24 h, and （4） plasmid transfection group,transfected with p47^phox siRNA for 24 h to block the NADPH oxidase protein subunit p47^phox in the HUVECs, or transfected with blank plasmid as control. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the mRNA and protein expression of p47^phox. The activation of extracellular signal-regulated kinase （ERK） and nuclear translocation of nuclear factor-κB （NF-κB）, stimu latory protein （SP）-I, and activator protein （AP）-I were assessed with Western blotting. Results The ROS level in the HUVECs of the TNF-α group was higher than that of the control group by 155.4%, and α- ZAL reduced the ROS level dose-dependently. TNF-α treatment up-regulated the p47^phox mRNA expression by 212. 8%, and obviously increased the p47^phox protein expression; and α-ZAL pretreatment attenuated the TNF-α-induced p47^phox mRNA expression by 63.0%, and also markedly inhibited the p47^phox protein expression. No obvious ROS was found in the HUVECs stimulated by TNF-α after the transfection of p47^phox siRNA. The ERK activation and nuclear translocation of transcription factors SP-1 and NF-κB induced by TNF-α were abolished or markedly inhibited by α-ZAL pretreatment. Conclusion α-ZAL has a potent inhibitory effect on the ROS production and ROS-activated signaling pathway in the TNF-α stimulated endothelial ceils, mainly through the inhibition of NADPH oxidase.